Tai Akera

A simple method for the determination of affinity and binding site concentration in receptor binding studies

In ligand binding studies, it is often difficult to apply kinetic analyses because of an uncertainty in experimental data obtained at high ligand concentrations. Under such circumstances, Kd value (an index of the affinity) and the binding site concentration may be estimated more accurately from the binding of a fixed concentration of labelled ligand observed in the presence of various concentrations of the non-labelled ligand, if the fraction of both labelled and non-labelled ligand bound is small. When there is no cooperative effect of the ligand binding, the Kd value may be calculated by subtracting the concentration of the labelled drug from the concentration of the non-labelled drug to cause a 50% reduction of the saturable binding of the labelled drug. From above values, the binding site concentration may be calculated. The proposed method is capable of examining the cooperativity of the ligand binding, the labelled drug concentration and the specific radioactivity of the labelled drug and does not require large amounts of the labelled drug.

Na+,K+-ATPase enzyme units in skeletal muscle from lean and obese mice

Abstract [ 3 H]-Ouabain binding to muscle preparations was utilized to estimate the number of Na + ,K + -ATPase enzyme units in hindlimbs from 8 week old lean and obese mice. Specific [ 3 H]-ouabain binding per mg particulate protein was 36% lower in obese mice; whereas, the affinity of the binding sites for ouabain was similar in obese and lean mice. Since obese mice had less muscle than lean mice, the number of Na + ,K + -ATPase enzyme units in hindlimbs from obese mice was less than half the number observed in lean mice.

Purification and characterization of the recombinant human aldose reductase expressed in baculovirus system.

Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.

Cardiac glycosides: Correlations among Na+, K+-ATPase, sodium pump and contractility in the guinea pig heart

SummaryRelationships among positive inotropic response to cardiac glycosides, Na+,K+-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na+,K+-ATPase activity was estimated from the initial velocity of (3H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive 86Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na+,K+-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na+,K+-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of 86Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na+,K+-ATPase and monovalent cation pump activities.

Quabain: Temporal relationship between the inotropic effect and the in vitro binding to, and dissociation from, (Na++K+)-activated ATPase

SummaryThe time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (3H)-ouabain binding to cardiac (Na++K+)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na++K+)-activated ATPase inhibition.Species differences were minimal either in the onset of inotropic response or the (3H)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in cat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species.Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na++K+)-activated ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.

Quantitative aspects of the interaction between ouabain and (Na+ + K+)-activated ATPase in vitro☆

Abstract The inhibitory effect of ouabain on (Na + + K + )-activated ATPase (Mg 2+ -dependent, (Na + + K + )-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na + , Mg 2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na + + K + )-activated ATPase reaction was subsequently started by the addition of a KCl solution. When the ouabain-enzyme reaction was brought to near equilibrium prior to the estimation of the ATPase activity, it was kinetically reversible, although overwhelmingly in favor of the ouabain-enzyme complex. This method resulted in a significant shift of the log concentration-response curve to the left. The concentration of ouabain to inhibit 50 % of the (Na + + K + )-activated ATPase activity was 0.12 μM, whereas it was 0.52 μM with the conventional method for the ATPase assay. Hence, the specific binding of ouabain to the (Na + + K + )-activated ATPase molecule was a slow process. This modified method was not suitable for the study of the effect of p -chloromercuribenzoate on (Na + + K + )-activated ATPase since the presence of ATP during the preincubation period protected the enzyme from the SH-blocking reagent. With the modified method, the effect of K + to antagonize ouabain inhibition of (Na + + K + )-activated ATPase was markedly reduced, indicating that the well-documented effect of K + was on the velocity of the ouabain-enzyme complex formation rather than on that of the ouabain-inhibited ATPase reaction. The ouabain-enzyme reaction was competitive with respect to K + at KCl concentrations below 5 mM, although the competition by K + was not remarkable. Above this concentration, the reaction was non-competitive with respect to K + . Ouabain released from the ouabain-enzyme complex was rebound to the enzyme during an incubation in the presence of Na + , Mg 2+ and ATP more easily than ouabain added to the incubation mixture.

Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.

Characterization of ultraviolet light-induced ouabain-resistant mutations in chinese hamster cells

Ouabain-resistant mutations in Chinese hamster cells have been quantitatively characterized. The mutation frequencies were found to be induced curvilinearly with treatments of increasing doses of ultraviolet light (UV). For the range of UV doses tested (5--20 J/m(2)), the observed frequency, Y, as a function of UV dose X, follows a curvilinear function, Y = (-28 + 13.37 X--1.52X(2) + 0.08X(3)) . 10(-6). The frequencies of UV-induced mutations were directly correlated with cell survival, indicating a similar causal relationship between cell killing and mutation induction. Under the same experimental conditions, X-rays induced 6--thioguanine-, but not ouabain-, resistant mutations. UV-induced ouabain-resistant (ouar) mutants exhibit a selection disadvantage. Their phenotypic expressions are modifiable by various agents. Wild type and 16 ouar mutants were compared with respect to their sensitivity to ouabain inhibition of 86Rb uptake by whole cells. All the ouar mutants assayed are less sensitive to the drug than are wild-type cells. In the absence of ouabain, the Na+--K+--ATPase activities can be significantly higher or lower than that of the wild-type cells.

Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase.

The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.

Amiloride: effects on myocardial force of contraction, sodium pump and Na+/Ca2+ exchange

The direct effects of amiloride on myocardial contractility were examined in electrically stimulated left atrial muscle of guinea-pig heart. Amiloride (0.3 to 1.5 mM) produced a positive inotropic effect which, at higher concentrations, was followed by a decline in developed tension. These effects were not accompanied by contracture or arrhythmia and were not affected by a combination of phentolamine, nadolol, cimetidine, tripelennamine and atropine. The above concentrations of amiloride prolonged the action potential duration during the development of the positive inotropic effect; however, no further change in the action potential duration was observed during the decline in developed tension caused by high concentrations of amiloride. Myocardial membrane Na,K-ATPase, ouabain-sensitive 86Rb+ uptake and Na+-dependent Ca2+ efflux from sarcolemmal membrane vesicles were all inhibited by amiloride. The positive inotropic effect of the agent is reduced and the negative inotropic action is enhanced in low Na+ solutions, i.e., under conditions likely to favor Ca2+ influx via Na+/Ca2+ exchange. These results suggest that amiloride, under the present conditions, has a complex interaction with cardiac muscle fibers. Amiloride may produce its inotropic effects in guinea-pig atrial muscle by several mechanisms including sodium pump inhibition, Na+/Ca2+ exchange inhibition, prolongation of the action potential duration, and/or actions such as Na+/H+ exchange inhibition which were not directly addressed in this study.